The development of therapies to eliminate or reduce the pool of cells latently infected with HIV-1 requires standardized assays that reliably and reproducibly assess the size of the latent reservoir. Three major challenges face the establishment of such assay: i) the very low frequency of latently infected cells; ii) the much greater frequency of cells harboring a defective over replication competent provirus; and iii) the lack of distinctive biomarkers of latently infected cells. So far, two approaches have been used to estimate the size of the latent reservoir. PCR-based assays measure HIV-1 DNA in a rapid, sensitive, and reproducible fashion, but cannot discriminate between replication competent and defective proviruses, and thus overestimates the reservoir. By converse, viral outgrowth assays (VOA's) determine the frequency of cells harboring replication competent provirus, but they are time and labor intensive, poorly reproducible, and do not capture the full extent of replication competent proviruses, leading to a significant underestimation of the reservoir. Our long-term goal is to develop technologies that combine the advantages of PCR with those of the QVOA into an assay that can be adapted to a single-cell, high-throughput platform. Our central hypothesis is that immune-PCR (iPCR) is the technology that makes that possible. The underlying principle of iPCR is the capture of an antigen of interest with a specific antibody, followed by detection with a second antigen-specific antibody tethered to a reporter DNA that is amplified in PCR reactions employing fluorescent probes for quantitative real-time analyses, as well as for single-cell in situ analyses. This project will benefit from the collaboration with Dr. Niel Constantine at the Institute of Human Virology in Baltimore, who developed and optimized the iPCR technology. In Specific Aim 1, we will develop a VOA where detection of p24 is carried out by iPCR, significantly improving sensitivity, reproducibility and practical performance of this assay. Detection of viral production by iPCR will be compared to other methods that estimate the size of the latent reservoir, both with and without viral expansion by co-culture with lymphoblasts of HIV-1 negative donors. In Specific Aim 2, we will develop techniques for ultrasensitive in situ detection of viral antigen production by iPCR and analysis by high-throughput flow cytometry. This approach will allow the identification and phenotypic characterization of single cells in which production of viral protein can be induced. Finally, in Specific Aim 3, we will evaluate whether iPCR can be used to achieve a more sensitive, rapid and reproducible detection of virologic effects of latency-reversing agents in ex vivo assays, and in vivo in clinical trials in which latency-reversing agents are administered to ART-treated HIV-1 patients. Accomplishing the goals of this project will fill the technological gap needed to obtain an accurate estimate of the latent reservoir in eradication trials, and a phenotypic characterization of latently infected cells.